Many gram positive bacteria, such as Mycobacterium tuberculosis produce ergothioneine (ESH) as protective small molecule thiol. 1,2,3 ESH is a thiohistidine betaine derivative with a thiol group at the C2 atom (ε-position) of the imidazole ring (Scheme 1). Recently, it was found that ESH is actively secreted into culture media by Mycobacterium smegmatis4 and present knowledge indicates that ESH may play a critical role in the in vivo and in vitro survival of mycobacteria. 
A structural variant of ESH, ovothiol A, also serves as an anti-oxidant albeit in sea urchin eggs as well as in the pathogens, Leishmania major and Trypanosoma cruzi.5 
Humans do not synthesize ESH, but possess an active transport system, a cation transporter (OCTN1) with high specificity for its uptake from dietary sources.6,7 
In 1956, Heath et al elucidated ESH biosynthesis in Claviceps purpurea. He demonstrated that histidine or a compound closely related to histidine might be a precursor of ESH, and subsequent publications disclosed the biosynthetic assembly of ESH utilizing organisms such as Neurospora crassa and Mycobacterium smegmatis with the aid of radio isotopic labelling (14C and 35S).8,9,10 
Melville et al further established the participation of the S-(β-amino-β-carboxyethyl)ergothioneine sulfoxide as an intermediate in ESH synthesis by incubation of hercynine in cell-free extracts of Neurospora crassa in the presence of O2 and Fe2+.11 
The sulfoxide is the substrate for the mycobacterial enzyme, EgtE. However, the absolute chirality of the sulfoxide is not known for the natural substrate or the synthetic one. Prior synthesis of intermediate (II) was reported in 1974 but was elaborate and irreproducible, and resulted in a low overall yield of 8.5%.15 The authors reported only the position of the aromatic proton resonance and no further structural confirmation. An optical rotation, [α]D +74.4 (c=0.5, H2O), was reported and could not be reconciled with the authentic natural product [α]D +9.1 (c=0.5, H2O). However, the m.p. of both natural and synthetic product was recorded as 188-190° C. None-the-less, it was claimed that synthetic S-(β-amino-β-carboxyethyl)ergothioneine sulfoxide (II) was extensively cleaved to ESH by crude cell free extracts of Neurospora Crassa. 
It has now been established that ESH is synthesized by the sequential action of five enzymes, encoded by the genes egtA, egtB, egtC, egtD and egtE (Scheme 1).12 EgtA is considered to be a γ-glutamyl cysteine ligase and catalyzes the formation of γ-glutamylcysteine. Histidine is methylated by an S-adenosylmethionine (SAM) dependant methyl transferase, EgtD, to give the trimethyl ammonium betaine, hercynine. Hercynine is then converted into S-(β-amino-β-carboxyethyl)ergothioneine sulfoxide (II), via an iron (II)-dependent oxidase (EgtB) which requires oxygen and γ-glutamylcysteine to produce γ-glutamylcysteinylhercynine (I). The exact nature of the latter transformation, in particular the sulfoxide formation, is still under investigation. Subsequently, a putative class-II glutamine amidotransamidase, EgtC, mediates the hydrolysis of the N-terminus glutamic acid, providing S-(β-amino-β-carboxyethyl)ergothioneine sulfoxide (II). Finally, EgtE, a pyridoxal 5-phosphate (PLP)-dependent β-lyase gives the final product, ESH.
Recently, the research focus with regard to these mercapohistidines has shed light on the mechanism of C—S bond formation at the δ- or ε-positions of the imidazole ring.13 OvoA is an iron (II) dependent sulfoxide synthase which catalyzes the first step in ovothiol A synthesis and is a homolog of EgtB. Interestingly, the substrate specificity of EgtB vs. OvoA in achieving C—S bond formation differs significantly. OvoA is very selective for its sulfur donor substrate and only accept L-cysteine while it prefers histidine as co-substrate. However, EgtB require γ-glutamyl-L-cysteine as sulfur donor. Furthermore, it is selective toward α-N,N,N-methylation on the histidine, i.e. hercynine as co-substrate. Surprisingly, OvoA switches its sulfurization pattern on the histidine ring from the δ-carbon to the ε-carbon depending on the level of α-N-methylation.14 Thus, OvoA converts hercynine directly into S-(β-amino-β-carboxyethyl)ergothioneine sulfoxide (II) and produces a minor amount of the δ-sulfoxide (ovothiol substitution pattern) when α-N,N-dimethyl histidine is used as the co-substrate (Scheme 1).

While the enzymes EgtB and EgtC have been expressed in a functional form, EgtE is still elusive and none of the enzymes have been thoroughly studied due to the lack of readily available substrate intermediates.
Recent commercial interest in ESH as a super anti-oxidant molecule has added even greater value to synthetic process development of this molecule. However, known synthetic processes for synthesizing ESH have only been able to achieve low to moderate yields at a very high cost. There is thus still a need to improve the process for synthetically producing ESH.